Bacterial foodborne illness and Escherichia coli O157:H7 strain infection among asymptomatic food handlers in Northeast Ethiopia: Implication for hygienic practices and mass‐screening

Abstract Background and Aims Food‐borne illness is a public health concern in developing countries because of improper food handling and sanitation practices, irregular medical checkups, lack of clean water supplies, and inadequate education among food handlers. This study investigated the burden of bacterial food‐borne illness, antibiotic resistance patterns, and associated factors among food handlers in prison and nonprison food establishment settings. Methods A cross‐sectional study was conducted from August 2022 to January 2023 among asymptomatic food handlers in Shewa Robit town. A total of 384 food handlers participated. Data were collected using structured questionnaires. Stool and hand swab samples were collected and cultivated onto MacConkey agar, xylose‐lysine‐deoxycholate, Mannitol salt agar, and blood agar, and incubated at 37°C. Bacterial species were identified using biochemical tests and gram staining. Mueller–Hinton agar was used in Kirby Bauer's disk diffusion method. Data were entered and analyzed using SPSS. Descriptive and logistic regression analysis were performed. Results Fecal and hand carriage rate of bacterial isolates were 106 (27.6%), and 214 (55.7%), respectively. Out of the 102 bacterial isolates, the most common ones from stool samples were Escherichia coli 71 (18.5%), Klebsiella aerogenes 12 (3.1%), and Salmonella spp. 7 (1.8%). Among 214 bacterial isolates, coagulase‐negative Staphylococci 115 (29.9%) and Staphylococci aureus 66 (17.3%) were identified from hand swab samples. Hand washing practice after restroom with water (adjusted odds ratio [AOR] = 2; 95% confidence interval [CI]: 1.16–3.45), irregular medical checkups (AOR = 2.49; 95% CI: 1.35–4.59), and did not receive food safety and hygiene training (AOR = 2.33; 95% CI: 1.34–4.05) were statistically significant association with food‐borne illness. Conclusions Foodborne pathogens pose a serious health risk in the study areas. The level of antimicrobial resistance are also concerning. Food handlers should therefore get strict regular health education, medical checkups, and training programs to prevent the spread of infections to the customers.

harmful, some, like O157: H7 strain, carry the exotoxin that can seriously poison humans who eat certain foods. 9 coli O157:H7 is one of the pathogenic serotype associated with severe diarrheal diseases that lead to hemorrhagic colitis, hemolytic uremic syndrome, and death, especially in immunocompromised individuals.The source of this bacterium is mainly food and animal foods in which the pathogen seeps into the food chain from improper hygienic practices either during the slaughtering of the animal or during food processing and servicing.10 One study from food of bovine origin in Ethiopia reported that the prevalence rates of E. coli O157:H7 was 6.5%.11 According to various reports, antimicrobial resistance (AMR) poses a serious threat to public health everywhere, 12 including in Ethiopia with different AMR reports among humans and animal products.11,13,14 Human can contract resistant bacteria from a variety of sources such as food products, the environment, and food handlers.A significant issue in developing countries is the emergence and spread of antibiotic-resistant bacteria, which can be easily contracted as a result of substandard living conditions and limited access to medical care.15 The frequency of food-borne illness, and E. coli O157:H7 infection distribution can be attributed due to variations on food production methods, inadequate food storage facilities, unsanitary food handling practices, resource constraints, and inadequate enforcement of regulatory norms.16 Few previous studies in Ethiopia focused on selected bacterial species such as Salmonella and Shigella, and intestinal parasites distribution among food handlers instead of all the bacterial pathogens causing food-borne illness.Therefore, this study investigated the prevalence of food-borne illness, E. coli O157:H7 strains, antibiotic resistance patterns, and associated factors among asymptomatic food handlers in Northeast Ethiopia.

| Study design, period, and settings
A cross-sectional study was carried out in both prison and nonprison settings in Shewa Robit town between August, 2022 and January, 2023.The Shewa Robit town is located in North Shewa Zone, Amhara Regional State, and 220 km from the capital city, Addis Ababa.An estimated of 54,307 people are living in the town according to the 2007 Central Statistical Agency census data.A total of 145 food handlers were employed in.Based on the Shewa Robit culture and Tourism Bureau information, there were a total of 16 hotels (with 211 food handlers), seven cafes (with 79 hand handlers), and 16 restaurants (with 90 food handlers).A source population consisted of all food handlers who were employed in Shewa Robit prison and nonprison settings.Food handlers who were actively involved in the preparation, handling, processing, or serving of food in selected nonprison settings and Shewa Robit Federal prison during the study period, as a study population.

| Sample size and sampling technique
Using a single population proportion formula, the final sample size was determined by taking into account 50% prevalence, 5% margin of error (d = 0.05), and 95% confidence interval (z = 1.96); n = (Zα/2) 2 p(1-p)/d 2 .Thus, 384 study participants were recruited and participated in this study.Food establishment setting including 106 food handlers from prison setting, 66 from restaurants, 154 from hotels, and 58 from café.Food handlers were selected using a simple random sampling (lottery method) technique in each study setting.

| Inclusions and exclusions criteria
All selected asymptomatic food handlers who were working in prison setting, hotels, cafés, and restaurants in Shewa Robit town were included in the study.Food handlers with clinical symptoms such as headache, nausea, vomiting, coughing, and diarrhea, and those who took antibiotics for 2 weeks before the study were excluded from the study.

| Demographic and hygienic practices data collection
Structured questionnaires comprising demographic and related factors (eight independent variables) and food hygiene practices (13 independent variables) data were collected.The questionnaires were pretested and validated before the actual data collection.The independent variables were socio-demographic information such as age, sex, marital status, level of education, monthly income, job position, service year, and hand washing habits before preparing food, hand washing after restroom with soap or water, cleaning equipment, fingernail status, training experience, status of medical checkups, wearing an apron and hair covering, and knowledge of food-borne illnesses.Selected food handlers were interviewed faceto-face by trained nurses.Continuous supervision, monitoring, and daily checks for data completeness were used to guarantee data quality.Data collectors were trained on how to collect data and samples from study participants.

| Samples collection, transportation, and processing
Using a clean, dry, and leak-proof stool cup, 2 g of stool samples were taken from each food handler.The specimens were put into the Cary-Blair transport medium, and transported to Debre Birhan compressive specialized hospital laboratory for laboratory analysis.Additionally, sterile cotton-tipped swabs dampened with normal saline were used to collect hand swabs from the palms of both hands of each food handler, and Amiens transport media was used to transport the samples. 176 | Bacterial culture, isolation, and identification Wire loop was used to streak samples or microbial cultures onto the surface of solid culture media plates.Samples were inoculated on culture media using zig-zag streaking technique (quadrant streak plating) to identify pure colony.Using a sterile wire loop, a stool sample was directly inoculated onto xylose-lysine-deoxycholate (XLD) and MacConkey (MAC) agar plates.The inoculated culture media was incubated at 37°C for 18-24 h, and examined for bacterial growth.The bacterial growth was detected and identified by colonial morphology and pigmentation, Simmons citrate, indole formation oxidase test, H 2 S production, urease production, and citrate utilization.18 Additionally, the swab sample was cultivated onto blood agar plate (BAP), MAC, and XLD, and Mannitol salt agar, and incubated at 37°C for 18-24 h.Standard biochemical tests including indole synthesis, Simmons citrate, urease, motility test, lysine, gas and sulfur production, glucose, and lactose fermentation, were used to detect and identify the bacterial species.19

| Identification of E. coli O157:H7 strain
The E. coli strains were differentiated using Sorbitol MacConkey Agar.
When E. coli O157:H7 was grown on MAC with Sorbitol and incubated aerobically at 37°C for 18-24 h.

| Data management and analysis
Data were entered into Epidata software and exported into SPSS version 26 for analysis.Descriptive statistics and logistic regression analyses were used.Model fitness was checked using the Hosmer-Lemeshow test.
Bivariable logistic regression was computed and variables with a p-value ≤ 0.2 were entered into the multivariable logistic regression analysis to control confounding variables.The crude and adjusted odds ratio were computed and p-value < 0.05 was considered statistically significant.

| OPERATIONAL DEFINITIONS
Food handler: is a person who directly engages in the preparation, processing, handling, and serving of food in the food establishment settings.
Nonprison setting: Food facility settings such as hotels, restaurants, and café in Shewa Robit town but not include prison setting.
Multiple drug resistance (MDR): Non-susceptibility to at least one or more antimicrobial agent in three or more antimicrobial categories.

| Socio-demographic characteristics of study participants
A total of 384 study participants were enrolled in the study.The study participants ranged in age from 16 to 42 years old, with a mean age of 24 years (SD ± 4.5).Among 384 study participants, the majority of study participants including 289 (75.3%) females, 252 (65.6%) waiters, 231 (60.2%) single participants, and 232 (60.4%) enrolled in the primary school (Table 1).

| Hygiene practice of food handlers
Many food handlers stated that they wash their hands with water alone (66.5%).Three hundred twenty-eight (85.4%) and 346 (90.1%) food handlers reported that they always wash their hands before preparing food and after using restroom, respectively.But, 141 (36.7%) of them made it a routine to wash their hands after handling unclean objects.Out of the total participants, only five (1.3%) of the participants have a certificate in food safety and hygiene.A total of 112 (29.2%) food handlers received in-service training on safe food preparation and handling practices (Table 2).

| MDR pattern of bacterial isolates
The overall MDR rate of this study was 17.9% (316/177).Of the detected bacterial isolates from fecal and hand samples, 58 (56.9%) and 121 (56.5%) bacteria had MDR levels.Salmonella species and K.
aerogenes showed comparatively higher rate of MDR, with average resistance of 71.4% and 91.6%, respectively (Tables 5 and 6).

| Associated factors with hand carriage of bacterial isolates
Out of all the associated factors evaluated in this study, hand washing practices of food handlers after restroom just water had a significant association (adjusted odds ratio [AOR] = 2.03, 95% confidence interval [CI]: 1.26-3.27,p < 0.001) with hand bacterial colonization compared to the counterpart (Table 7).Foodborne illness is a leading cause of illness and mortality worldwide.

| Associated factors with fecal carriage bacteria isolates
Food handlers may put customers at risk of bacterial infection. 21ong asymptomatic food handlers, the rate of bacterial carriage in feces and hands were 102 (26.7%), and 214 (55.7%), respectively.The rate of bacterial fecal carriage was 102 (26.7%), greater than the reports in Gondar (13.2%), 22 Jimma (6.9%), 23 and Thailand (2.5%). 24reover, the prevalence of food-borne pathogens among food The majority of bacteria found in the fecal samples was E. coli (18.5%) which is consistent with a study in Gondar that found E. coli to be prevalent. 25However, a study reported in Jimma indicated that Salmonella species (40%) was predominant. 23The prevalence of E. coli O157:H7 strains was 4.2%, which is lower compared to studies done in Southern Ethiopia (26%) 26 and Nepal (10.3%). 27This finding was also consistent with a study conducted in Ethiopia (1.8%). 23In addition, the prevalence of fecal Salmonella species was 1.8%, which is consistent with a study reported in Southern Ethiopia (2.1%). 28wever, the prevalence of fecal Salmonella species in this study was lower than studies documented in Northwest Ethiopia (4.1%), 29 and Addis Ababa (3.5%), 5 Sudan (4.4%), 30 and Pakistan (9%). 31ere was no fecal Shigella species identified among food handlers, which is similar with a finding in Southern Ethiopia. 28The hand carriage rate of bacteria was 56.2% in this study, which is higher than a study done in Debre Markos (29.5%). 32Our findings was also lower than studies reported in Gondar (70%), 33 and Iran (62.2%). 34In this study, coagulase-negative staphylococci (29.9%) was predominant followed by S. aureus (17.2%) and E. coli (6.8%).These findings are similar with a study documented in Saudi Arabia (39.9% vs. 17.5%). 35e hand carriage rate of E. coli was 6.8%, which is similar with a study in Jimma (10.9%) 36 but lower than a study conducted in Nigeria (1.8%). 35The discrepancies might be due to variations of bacterial identification techniques, personal hygiene, hygiene practices during food processing and handling, and study settings.Good food safety practice is essential for the prevention and control of foodborne illness and it is a priority concern to protect the health of the community.sulfamethoxazole (33.3%), ciprofloxacin (14.2%), and chloramphenicol (3.9%). 37and Morocco, reports as the highest resistance rates were shown against ampicillin and amoxicillin-clavulanate acid (100%), and cefotaxime (60%). 38This study also indicated that there is a concern on the rate of E. coli O157:H7 strains, accounting 4.2%.

E. coli
E. coli O157:H7 strains had chloramphenicol and trimethoprimsulfamethoxazole resistance each 50%, which is consistency with a study done in Nigeria, where the prevalence of chloramphenicol and trimethoprim-sulfamethoxazole resistance were 60.4% and 31.2%,respectively. 39This distribution of E. coli O157:H7 strains indicated that there is a poor food hygiene practices and its transmission to the customers, which is also supported in the associated factors analysis in this study.The hot environment of Shiwa Robit town may pique the interest of food handlers during food handling and processing, which results to poor food hygiene practices.This results that the consumers served in these food outlets are at risk of developing food-borne illness and E. coli O157:H7 infections.
The results of our study revealed that the S. aureus isolates were resistant to trimethoprim-sulphamethoxazole (57.6%), ciprofloxacin (49.9%), and gentamycin (49.9%).This report was significantly less than that of the study that was conducted in Debre Markos, Ethiopia, which involved ciprofloxacin (9.1%) and trimethoprimsulphamethoxazole (18.2%). 32Pseudomonas resistant to gentamycin (100%) and ciprofloxacin (100%) in hand swab samples which showed higher levels of resistance to both antibiotics (ciprofloxacin [33%], gentamycin [33%]) than a study carried out in Debre Markos, Ethiopia. 32lmonella species isolated from fecal samples were highly resistant to trimethoprim-sulfamethoxazole (57.1%), and ciprofloxacin (42.9%), which is comparable to a study conducted in Dilla, Ethiopia, where the prevalence of ampicillin (81%) and amoxicillin-clavulanic acid (>70%) resistance was seen. 40Our finding was higher than a study reported in Hawassa, Ethiopia, where the prevalence of tetracycline, ampicillin, and trimethoprim-sulfamethoxazole resistance was 80%, 60%, and 40%, respectively. 28The blind prescription medications in the clinical domain may be the cause of this high level antibiotic resistance.As a result, there will be no alternative medications available to treat highly drug-resistant strains.
According to the results of the current study, Pseudomonas from hand swab samples exhibit 100% resistant to both gentamycin and ciprofloxacin, which is higher than the result of study in Debre Markos, Ethiopia, where both ciprofloxacin and gentamycin resistance were found to be 33%. 32This finding indicated that MDR bacteria accounted for 18% of the bacterial isolates.An increase in MDR could be attributed to the improper and inappropriate use of antimicrobial drugs as a result of empirical treatment and inadequate infection prevention and control measures, such as environmental cleaning and hand hygiene.
Interesting findings have been made about the association between hand washing practice with just water and bacterial colonization.Compared to hand washing with soap, the practice of hand washing after using the restroom with just water was strongly linked with bacterial colonization.This finding was supported by previous studies in both Adigrat and Arba Minch, Ethiopia. 41,42In fact, washing hands with soap is more effective than with water alone because the surfactants in soap help to remove dirty and bacteria from the skin, and individuals prefer to wash their hands more thoroughly with soap, which helps to further eliminate germs.
Those food handlers who had an irregular medical checkups were significantly associated with bacterial colonization compared to the counterparts.A study conducted in Dilla, Ethiopia is comparable to this one. 40This might be due to the fact that food handlers who examined their health status regularly had a better understanding about safe food handling and processing.In addition, they also get professional advices during medical cheek-ups which enhances their overall performance in safe food handling practices.Food handlers who didn't receive food safety and hygiene training were 2.33 times more likely to be colonize by bacteria compared to the counterparts.This finding also supported by a study conducted in Jimma, Ethiopia. 23Food handlers who were properly trained can take the necessary precautions to avoid malpractice during food handling and safety.A meta-analysis study in Ethiopia supports this study which stated that food handlers with formal education, good knowledge, received training, and a positive attitude about food hygiene components, as well as regular medical checkups were significantly associated with good food hygiene practice. 43Therefore, food handlers should attend proper training in the basic principle of food safety and rules of personal hygiene to improve their practices during food handling.
This study have some limitation such as the nature of cross-sectional study which could not assess temporality of the cause-effect relationships, and recall bias for some questions among participants.

| CONCLUSIONS
The carriage rate of bacteria and their drug resistance among asymptomatic food handlers are a priority.E. coli, S. aureus, and CONS were the most predominant bacteria identified from food handlers, and the more risky E. coli O157:H7 strain was identified.
The rate of MDR of bacteria was 18%.This indicates that food handlers may transmit food-borne disease to the consumers including drug-resistant bacteria.Hand washing practices after restroom with water alone, irregular medical checkups, and do not receive food safety and hygiene training were significantly associated with bacterial colonization.We recommend that food handlers undertake regular medical checkups, health education, and continuous food safety and hygienic practice trainings to prevent the transmission of food-borne diseases to the customers and the community at large.
Ethical approval was obtained from the Research and Ethics Review Committee of College of Medicine and Health Sciences, Wollo University (Ref: CMHS/12/2022).A formal written letter was obtained from the Shewa Robit city administration health office and the prison authorities.Written informed consent was obtained from each study participant before data collection and participated voluntarily.The purpose and procedures of the study were explained to each study participant during data collection.Positive results were communicated to the study participant, prison health officials, and Shewa Robit city administration health office for further assessment and treatment.The confidentiality was maintained.
characteristics of food handlers in prison and nonprison food establishment settings in Shewa Robit town (N = 384).
Food hygiene practices of food handlers at prison and nonprison food establishment settings in Shewa Robit town (N = 384).
irregular medical checkups (AOR = 2.49, 95% CI: 1.35-4.59,p<0.001), did not receive food safety and hygiene training (AOR = 2.33, 95% CI: 1.34-4.05,p<0.001), and hand washing practice after restroom with just water (AOR = 2, 95% CI: 1.16-3.45,p<0.001) were significantly associated with fecal bacterial colonization (Table8).T A B L E 2 F I G U R E 1 Hand carriage rate of bacterial contamination among food handlers in prison and nonprison food establishment settings in Shewa Robit town.F I G U R E 2 Fecal carriage rate of bacterial species among food handlers at prison and nonprison food establishment settings in Shewa Robit town.
Antimicrobial resistance patterns of bacterial isolates detected from hand swabs among food handlers at prison and nonprison food establishment settings in Shewa Robit town (N = 384).
and 34.9% (37/106; from fecal samples).This finding may indicates that the prisoners are at risk and more vulnerable compared to the general population for acquisition of food-borne illness due to limited washing facilities, inadequate and clean water supplies which results poor personal hygiene, and overcrowding nature of the prisoners living in one prison houses/cells in Ethiopia.T A B L E 3 Antimicrobial resistance patterns of fecal-bacterial isolates among food handlers at prison and nonprison food establishment settings in Shewa Robit town.
Multidrug resistance (MDR) patterns of fecal bacterial isolates detected from food handlers at prison and nonprison food establishment settings in Shewa Robit town.Multidrug resistance (MDR) patterns of bacterial isolates detected from hands of food handlers at prison and nonprison food establishment settings in Shewa Robit town.Associated factors for acquisition of bacterial hand colonization using bivariate and multivariate analysis among food handlers in prison and nonprison food establishment settings in Shewa Robit town.Bivariate and multivariate analysis to identify associated factors with fecal carriage of bacteria among food handlers in prison and nonprison food establishment settings in Shewa Robit town.
*Significant variables at p < 0.05.T A B L E 8*Significant variables at p < 0.05.